Project Summary The development and spread of antibiotic resistance is jeopardizing the efficacy of our current antibiotic arsenal. To formulate targeted therapies that make better use of existing antibiotics and reduce the development of resistance, we must understand how antibiotics impact both the pathogenic and beneficial members of the human microbiome. This is particularly important because the disruption of microbiome homeostasis by antibiotics is associated with multiple microbiome-related diseases, such as Clostridium difficile-associated diarrhea and inflammatory bowel disease. Current descriptive microbiome research, focused on identifying taxonomic changes, has not addressed the mechanistic question of why specific bacteria within a microbial community are negatively impacted by antibiotics while others are not. The work proposed here will move past this limitation by transcriptionally profiling the impacts of antibiotics on the total microbial community in vivo to provide functional and mechanistic insight into the action of antibiotics in the microbiome. The central hypothesis of this study is that the induction of tolerance and resistance mechanisms mediates toxicity to antibiotic exposure in susceptible members of the microbiome. This proposal is focused on tolerance mechanisms related to the metabolic state of bacteria before and during treatment. The microbiome consists of many metabolic microenvironments and within these communities metabolically active bacteria are likely to be more susceptible than less active species. Total transcriptional profiles of the microbiome can be used to study the roles of well- defined tolerance and resistance mechanisms within the microbiome in vivo. This project will test our central hypothesis in three aims: Aim 1) Determine the impacts of broad-spectrum antibiotics on the structure of the salivary microbiome derived from clinical samples. Aim 2) Determine the total transcriptional response of the microbiome to bactericidal and bacteriostatic antibiotic therapy. Aim 3) Profile the impacts of ciprofloxacin on the structure and function of the murine microbiome in conjunction with host metabolic perturbation. In addition to testing this hypothesis, a key goal of this exploratory proposal is to implement a novel outpatient-based methodology to study the response of the human microbiome to antibiotic therapy. The ultimate goal of this work is to provide information about the impact of antibiotic therapy on the structure and function of the microbiome, in order to allow clinicians to select therapies that minimize microbiome-related complications and the transfer and development of resistance. This basic knowledge will help clinicians to improve antibiotic therapy by promoting evidence-based, targeted treatments to safeguard our current arsenal of antibiotics.